Tissue High Throughput Screening Platforms. The failure of promising drug-like compounds to successfully complete clinical trials, despite our ever-increasing knowledge of genomics, proteomics, and cell signaling events in the basic medical sciences suggests that the preclinical models scientists are using in academia and industry alike are not predictive of the overall drug efficacy and safety. Most cell-based systems for high throughput screening (HTS) are cultured in a monolayer, despite the known importance of the cell and tissue microenvironment. Studies in breast tumor cells have highlighted the importance of culturing cells in 3-dimensions (3D), with cell-cell and stromal interactions. The cellular context and environment provide signals critical to a cell's fate. As such, it is recognized that 3D models (aka spheroids) may be a more predictive preclinical model. However, technical and financial hurdles prevent 3D models from meeting their promise in current HTS efforts. In addition, much of what makes 3D cultures interesting lies in the inherent architecture where the inner and outer cells vary with respect to growth, nutrient access, hypoxia, and sensitivity to drugs. Thus, there is a true, unmet need for new 3D culture systems in plug and play modules that can work with multiple cell types, is cost-effective, and can be utilized by anyone doing HTS to improve their drug discovery efforts, especially with respect to finding drugs that will translate to the clinic and act safely in patients. We propose that human cells cultured in a multi-well format as multicellular spheroids can bridge that gap. We have successfully cultured various human cancer cell lines as 3D spheroids in a 96-well format amenable for HTS. Under the appropriate conditions, 1 spheroid forms per well, and Z'factors of greater than 0.5 have been achieved in proof-of-concept assays which compared 3D to 2D culture conditions. In our preliminary screens, anti-tumor compounds have been identified which exhibit differential effects in the cancer cells cultured as 2D monolayers versus those in 3D. Here we propose to further our ability to model the tumor microenvironment by introducing human endothelial cells and fibroblasts to the human cancer cells to form 3D co-cultures. Clearly, tumors are not composed of one cell type, and all the cells of the tumor contribute to the overall response to anti-tumor therapy. In addition, we will step away from traditional cell culture medium formulations and techniques and instead culture the cells under physiological glucose, glutamine, and oxygen concentrations to mimic the human body. Next, we will develop 3D co-cultures using normal human cells as a model for drug safety. Lastly, we will perform a proof of concept HTS using these new models. The resulting protocols for the co-culture of epithelial, endothelial, and stromal cells for HTS to test efficacy or safety will be published in the public domain, and any leads will be further validated using high content screening to visualize the cells in their 3D context.